The cell cycle is regulated by the activities of cyclin-dependent kinases (Cdks). In mammals, 11 different Cdks have been identified whereas in yeast only one major Cdk drives the cell cycle. Of the 11 mammalian Cdks, Cdk4 and Cdk6 promote entry and progression through G1, Cdk2 functions in entry and progression through S-phase, Cdc2 (Cdk1) regulates M-phase, and the other Cdks function in transcription or have unknown functions. The activities of Cdks are regulated by protein-protein interactions (with cyclins, inhibitors, and assembly factors), protein degradation, transcriptional control, subcellular localization, and multiple phosphorylations. Several functions for Cdk2 have been suggested, including entry into S-phase by Rb phosphorylation, initiation of DNA replication, exit from S-phase, and progression through G2 phase. We have generated Cdk2 knockout mice and found that they are viable but sterile. Our results demonstrate that Cdk2 is essential for meiosis but not mitosis in vivo.- Aim 1:Functions of Cdk2 during tumorigenesis.- Aim 2:Genetic and biochemical analysis of Cdk2 pathways.- Aim 3:Analysis of the Cdc2 locus in the mouse- Aim 3:Regulation of CDK6/KSHV-cyclin complexes.Aim 1:Functions of Cdk2 during tumorigenesis.In tumors, the regulation of the cell cycle is altered to allow for increased proliferation. We are now studying the loss of Cdk2 during tumorigenesis. Four different models will be employed: (1) chemically induced skin tumor model, (2) chemically induced liver tumor model, (3) gamma irradiation model, and (4) a loss of p53 induced model. For the skin tumor experiments (1), mice at 7 to 8 weeks of age were shaved and up to 400 nmoles of 9,10,-Dimethyl-1,2-Benzanthracene DMBA were painted on the skin. Starting 10 to 20 days later twice weekly doses of up to 10 nmoles of 12-O-tetradecanoylphorbol-13-acetate TPA was painted on the shaved areas. Papillomas appeared between 7 to 10 weeks after the first dose of TPA. After 20 weeks, the number of papillomas per mouse averaged between 10 and 15, which were collected along with normal skin samples after the mice were euthanized. All skin samples will be analyzed for genetic mutations in the Ras gene by PCR and at the same time protein levels of cell cycle regulators will be investigated by Western blots or immunohistochemistry. We have done a pilot experiment and are currently repeating it with a larger cohort of mice. Our results indicate that Cdk2-/- mice develop less papillomas than wild type mice and therefore are less susceptible to skin tumors. In order to study if effect of Cdk2-/- is specific to skin tumors, we will investigate other tumor models in the Cdk2-/- background. We will use a liver tumor model, gamma irradiation, and a p53 model. We have generated double mutant mice lacking Cdk2 and p53 and these mice are viable. So far, we have only few animals and we will start the analysis of these mice soon.All these experiments aim to investigate the efficiency of cancer therapy by inhibiting Cdk2. This is a very important link between our experiments and therapeutical intervention.Aim 2:Genetic and biochemical analysis of Cdk2 pathways.Cdk2-/- mice did not display a sever phenotype. Therefore, we have to define the pathways in which Cdk2 is involved in more details. We are approaching these questions by mouse genetics and biochemistry. Double knockout mice of Cdk2 and Cdk4 or p27 are being generated at this moment. So far we have determined that Cdk2-/-p27-/- double mutants are viable and that these mice display all the p27-/- as well as Cdk2-/- phenotypes. Therefore, Cdk2 did not rescue the p27-/- phenotypes and there must be other targets of p27. We have identified Cdc2 are an important target of p27.We aimed to generate Cdk2-/-Cdk4-/- mice, but this is quite difficult since both genes are on the same chromosome and only 1 cM apart. After screening a large number of offspring, we have identified two animals that had Cdk2 and Cdk4 mutations on the same chromosome cis. Now we are generating homozygous double mutant and should have offspring in a few weeks. In the mean time, we are analyzing mouse embryonic fibroblasts MEFs from Cdk2-/- embryos where Cdk4 was silenced by shRNA technique. Such cells are able to proliferate and we are analyzing them in detail.Aim 3:Analysis of the Cdc2 locus in the mouseWe are also interested in the functions of Cdc2, since in the absence of Cdk2, Cdc2 might compensate for its functions. This is especially important since we have found that the cyclin A functions are not impaired in the absence of Cdk2 and this is due to Cdc2 binding. Therefore, we are generating conditional Cdc2 knockout mice and knocking-in Cdk2 into the Cdc2 locus. Currently, the targeting vector for the knockin mice is finished and we will generate the mice within 6 months.These mice will be the basis for the biochemical analysis similar to the one that we are engaging for the analysis of the Cdk2-/- mice.Aim 4:Regulation of CDK6/KSHV-cyclin complexes.One step in the activation of Cdks is phosphorylation by the Cdk-activating kinase (CAK). We demonstrated the KSHV-cyclin could activate CDK6 in the absence of CAK phosphorylation in vitro and in vivo. A possible explanation is that CDK6 and/or KSHV-cyclin are phosphorylated at unknown sites. Therefore, we made mutations at potential phosphorylation sites. For KSHV-cyclin, we made more than 20 mutants and found in a first analysis that all of them are active in vitro and when transfected in cells. Interestingly, deletion of the first 19 or 45 amino acids of the KSHV-cyclin did not affect phosphorylation of the Retinoblastoma protein Rb but inhibited phosphorylation of histone H1. This effect is now further investigated by using a N-terminal peptide and also transferring this N-terminal KSHV-cyclin peptide to cyclin D1, which usually does not phosphorylate histone H1.A manuscript is in revision for the Journal of Biological Chemistry.